This invention relates in general to devices for separating crystals from urine or other biological fluids and ascertaining their chemical identity. Also provided are methods for detecting and identifying crystals which may be present in urine or other biological fluids, and kits containing the devices and other materials and reagents needed to conduct the methods.
Crystalluria, the presence of crystals in the urine, is a common problem which afflicts household pets such as cats and dogs. The formation of crystals and mucus in the urinary tract is a potentially life threatening condition in these animals. It is indicative of the predilection to form uroliths or stones, and can ultimately lead to a complete obstruction of the urinary system. Some studies have recorded that almost 10% of male cats and over 3% of female cats are affected by this disease.
Urine crystals may be of several different types. Most commonly crystals are formed of struvite (magnesium-ammonium-phosphate), oxalate, urate, cystine, or silicate, but may also be composed of other materials such as bilirubin, calcium carbonate, or calcium phosphate. Struvites and calcium oxalates combined comprise over 88% of the uroliths found in cats and dogs and therefore represent the cause of the great majority of these animal medical problems. The occurrence of crystalluria within these animal populations varies according to species, breed, diet, sex, age, and genetic pre-disposition. Crystalluria also occurs in a variety of other mammals. For example, it is known that calcium carbonate crystals can form in horses, rabbits, guinea pigs, and goats.
Present methods of detecting and identifying urine crystals rely on a high level of skill of a person who examines the crystals under a microscope and makes distinctions between the different types of crystals that may be present. It is sometimes required for the sample to be sent to a commercial laboratory for the analysis.
The term xe2x80x9chabitxe2x80x9d is commonly used by minerologists to refer to the characteristic shape or shapes of mineral crystals. Different crystal types exhibit different habits which a skilled person is able to differentiate. The judgment is based largely on the apparent structure or habit of the crystals. These methods involve specific skills, significant handling of the specimen, and are both time consuming and expensive. Microscopic detection and identification of the crystals is further complicated by the fact that their appearance can be influenced by the variable conditions of their formation, growth, and dissolution. The sensitivity of this method is also limited as it is not uncommon for the technician to fail to detect small numbers of crystals which may be present. This method of identifying the crystals also involves significant handling of the sample since it typically requires a volume of 5-10 mls of urine, which then must be concentrated to enhance the population of crystals within the examined specimen. Concentration is normally accomplished by centrifugation at 2500 rpm for 5-10 minutes, aspiration of all but 0.5 to 1.0 ml of fluid, and resuspension of the pellet.
The microscopist bases the judgment of crystal type on the physical characteristics or habit of the crystals present. Calcium oxalate dihydrate crystals typically are colorless and have a characteristic octahedral or envelope shape, having the appearance under a light microscope of squares whose corners are connected by intersecting diagonal lines. Struvite crystals are known for their colorless, orthorhombic, xe2x80x9ccoffinlidxe2x80x9d shape, although frequently other, irregular forms are seen. They often have three to six or more sides and often have oblique ends. Cystine crystals exhibit a colorless hexagonal shape with equal or unequal sides. They may appear singly but usually aggregate in layers. As is evident from these descriptions, the differentiation of these different crystal types is based largely on subjective criteria, and is therefore prone to human error. For example, skilled persons sometimes have difficulty distinguishing struvite crystals from cystine. Calcium carbonate crystals may form as large yellow-brown or colorless spheroids with radial striations, or smaller crystals with round, ovoid, or dumbbell shapes.
Furthermore, the outer appearance of a crystal may not always correlate with its true chemical identity. For example, kidney stones are usually comprised of calcium oxalate, struvite, or cystine. However, crystals sometimes are comprised of a calcium oxalate core covered by an outer layer of struvite. Similarly, they may be comprised of a struvite core covered by an outer layer of calcium oxalate. Therefore, such crystals can be very deceiving even to the skilled person who is trying to ascertain their identity based largely on the external appearance of the crystals. The distinction is important, since the treatment programs for struvite and calcium oxalate crystals are very different.
Furthermore, before existing chemical and enzymatic analyses can be performed on crystals from urine or other biological fluids, it is necessary to first remove certain materials which are normally present and which interfere with these reactions, such as free magnesium, oxalate, and other substances. These materials are removed because they are not indicative of the presence of crystals when they are in the dissolved state. Therefore the use of these methods involves the further inconvenience of having to remove small molecular weight interfering substances such as oxalates which are typically removed by activated charcoal prior to testing.
Because of the time and expense involved in making accurate determinations of the presence and type of crystals, many veterinarians simply measure the pH of a urine sample, and make prescriptive decisions based on this criteria alone. This often results in needless changes to the animal""s diet, and unnecessary inconvenience to the owner and stress on the animal.
The present invention provides a device for the convenient, rapid, and accurate determination of crystals which may be present in urine or other biological fluids and their identity. The device is inexpensive, disposable, specific, and requires only very small sample volumes. It also eliminates the need and consequent delay and expense of transmitting samples to a commercial laboratory for analysis. This results in quicker treatment for the affected animal and avoids additional stress on the animal caused by unnecessary dietary changes and inconvenience to the animal owner. Another important advantage is that the analysis of the urine crystals is based on more objective criteria, sharply reducing human error as a source of inaccuracy. Using the present invention, one is able to determine the presence of urinary crystals and identify the crystal type with almost 100% sensitivity and accuracy.
The present invention also discloses methods for detecting and identifying crystals which may be present in biological fluids, including urine. The methods may be conveniently performed in the veterinarian""s office during the time typically taken for an office visit. Unlike currently available methods, a high level of training and skill is not necessary to successfully and confidently identify the crystal types, and the method can be learned in a matter of minutes.
The present invention also discloses kits which may include the devices of the present invention and reagents necessary for conducting the methods of the present invention. The kits enable the veterinarian or other animal caretaker to have conveniently available everything needed to conduct the assay and detect and identify any crystals which may be present. The kits have a shelf life of at least six months and may last one year or more, and may be conveniently stored in a small space until needed. The kits may include reagents necessary to conduct the assay in a ready-to-use format, thereby eliminating the need for mixing or preparing reagents.
The present invention relates to devices, methods, and kits for the rapid detection and identification of crystals which are suspected of being present in urine or other biological fluids. The devices of the present invention contain a filter, and may also contain an absorbent material. Crystals which are suspected of being present in urine or other biological fluids are isolated on or within the filter. The crystals may be isolated on the surface of or within the structure of the filter. The absorbent material serves to facilitate the drawing of liquid through the filter and into its absorbent material, thus isolating the crystal on or within the structure of the filter. Once isolated, the crystals will be available for staining with an indicator reagent specific for a determining component of the crystal.
In a preferred embodiment, the device may further contain a member which inhibits flow from the absorbent material to the filter and which may be placed between the absorbent material and the filter. The filter, absorbent material, and member for inhibiting flow from the absorbent material to the filter may be in positional relationship such that fluid passes through the filter, through the member for inhibiting flow from the absorbent material to the filter, and into the absorbent material. The member for inhibiting flow from the filter to the absorbing material may be made of polyethylene. In a preferred embodiment, the member may be a disk of porous polyethylene.
In preferred embodiments, the filter may be a filter with a graded pore structure. In various embodiments, the filter may be selected from, but is not limited to, an A/E glass filter, glass fiber, mylar, WHATMAN D28(copyright) filter, WHATMAN GD-1(copyright) filter, nitrocellulose, HEMASEP V(copyright), HEMASEP L(copyright) filter, or SUPOR(copyright) polyethersulfone filter. The biological fluid may be urine, blood, blood products, or interstitial fluid.
In another aspect, the present invention provides methods of detecting and isolating crystals which may be present in urine or other biological fluids. The methods of the present invention may consist of the steps of isolating the crystals on or within a filter, contacting the crystals with an indicator reagent which is specific for a determining component of the crystals, and determining the presence and identity of the crystals.
The methods may further consist of a step of washing the crystals to wash away contaminants which may interfere with the indicator reaction. This wash step may be necessary in some chemistries to obtain an optimal indicator reaction. This washing step may further accomplish a disintegration action on the crystals thereby exposing a determining component of the crystals. There may be an additional washing step performed after the step of contacting the crystals with the indicator. This step may be combined with the initial wash step by using a wash that both washes and acts to partially disintegrate the crystal. The determining component thus exposed may react with dye or substrate for that specific determining component of the crystal. In preferred embodiments, the presence of the determining component will cause a visible color change on the surface of or within the filter or membrane. The additional wash step may clarify the color change for easier interpretation.
In preferred embodiments, the crystals which are suspected of being present may be oxalate crystals, or struvite crystals. The indicating system may be oxalate oxidase, a peroxidase and an indicator. In preferred embodiments, the peroxidase may be horseradish peroxidase. The indicator may also be a precipitating magnesium binding dye, such as magneson dye, or a calcium binding dye. In preferred embodiments, the indicator may be these or other precipitating dyes which form insoluble complexes with various analytes.
The present invention also provides kits for detecting and identifying crystals which are suspected of being present in urine or another biological fluid. The kits may include a device of the present invention for detecting and identifying crystals which are suspected of being present in a biological fluid and at least one reagent for detecting and identifying the crystals. The reagent(s) provided in the kit may be an indicator reagent or system useful for practicing the methods of the present invention. The kit may also include other reagents useful for practicing the methods of the present invention.